Fluorescence immunoassays using organo-metallic complexes for energy transfer

ABSTRACT

The invention includes a composition of matter and method that utilizes energy transfer between one or more donor and acceptor molecules. The composition of matter includes an encapsulation vesicle having a matrix, a surface coating of an organo-metallic complex and a transparent protection layer. The transparent protection layer is capable of modification by addition of biomolecules to the surface in order to bind other molecules. The proximity of the bound biomolecules to the protective layer allows for energy transfer from a donor molecule internal to the protection layer to an acceptor molecule outside the protection layer. The protection layer acts to diminish the effects of collisional quenching on the donor molecules caused by ubiquitous small molecules such as molecular oxygen. The application also teaches a method of making and applying the complexes to immunoassays.

FIELD OF THE INVENTION

[0001] This invention relates to fluorescence immunoassays, DNAhybridization assays, and more particularly to a composition of matterand method for using organo-metallic complexes for assays that can beeasily quenched by interfering molecules in energy transfer processes.

BACKGROUND OF THE INVENTION

[0002] Fluorescence immunoassays based on energy transfer have beentaught by Lakowicz et al. in U.S. Pat. No. 5,631,169. A number ofmolecular species have been used for causing energy transfer from adonor molecule to an acceptor molecule. In particular, sandwich typeimmuno-complex formation can be used with this technique. For instance,this technique can be used for immunoassays based on changes influorescence lifetime with changing analyte concentration. However, thistechnique works with short lifetime dyes like fluorescein isothiocyanate(FITC) (the donor) whose fluorescence is quenched by energy transfer toEosin (the acceptor). A problem or disadvantage of this technique isthat short lifetimes are extremely difficult to measure. Moreover, thedonors used in this invention have relatively short Stoke shifts, whichcould cause excitation signal to overlap the acceptor absorption therebycausing spurious signals, particularly when broad band emitters likeLEDs are used as excitation sources.

[0003] Organo-metallic complexes can be used as donors withoutidentifying suitable acceptors. However, there are a number of smallmolecules such as oxygen that also quench fluorescence of thesecomplexes. For these reasons a number of problems exist when using someof the standard organo-metallic complexes for quenching experiments,measurements or immunoassay studies. Organo-metallic complexes, however,have the advantage of providing more suitable long lifetime basedsensing and quenching. Consequently, assays based on this principlewould require that the sample be purged of oxygen prior to analysis.This not only adds an additional sample preparation step, but alsoprecludes one from analyzing samples that could be altered by suchprocessing.

[0004] Accordingly, there is a substantial need for techniques andcompositions of matter that allow for the use of a long lifetime donorsto be used in conjunction with immunoassays based on the use of energytransfer. In addition, there is a need for donors that will allow forlarger Stoke shifts, but not suffer from the limitation of quenching bymolecules such as molecular oxygen. Furthermore, there is a need fortechniques and compositions of matter that allow for the use oforgano-metallic complexes that can be applied in the presence ofchanging concentration of collisional quenchers.

[0005] The above reference(s) and all other references cited in thisapplication are incorporated in this application by reference. However,cited references or art are not admitted to be prior art to thisapplication.

SUMMARY OF THE INVENTION

[0006] The invention is a composition of matter and method of using thesame for immunoassays. The encapsulation vesicle comprises a matrix, asurface coating with an organo-metallic complex and a transparentprotection layer. The protection layer is capable of modification byaddition of biomolecules to the exterior surface. The biomolecules maycomprise one or more acceptor molecules. The proximity of the boundbiomolecules to the protection layer allows for energy transfer fromdonor molecules that are inside of the transparent protection layer tothe acceptor molecules that are outside the transparent protectionlayer. The transparent protection layer acts to diminish the effects ofcollisional quenching by small molecules such as oxygen to the donormolecules. The method includes a number of novel immunoassays thatutilizes energy transfer between one or more donor and acceptormolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Embodiments of the invention will now be described with referenceto the drawings in which:

[0008]FIG. 1 illustrates a first embodiment and cross-section of thecomposition of matter of the present invention.

[0009]FIG. 2 illustrates a second embodiment and cross-section of thecomposition of matter of the present invention with acceptor moleculesdirectly attached to the protection layer.

[0010]FIG. 3 shows a third embodiment of the composition of matter ofthe present invention with the acceptor molecules attached to a separatesubstrate.

[0011]FIG. 4A shows a first embodiment and method of the presentinvention using a standard immunoassay.

[0012]FIG. 4B shows a second embodiment and method of the presentinvention using a sandwich immunoassay.

[0013]FIG. 4C shows a third embodiment and method of the presentinvention using a nucleic acid probe.

[0014]FIG. 4D shows a fourth embodiment and method of the presentinvention using an aptamer and protein.

[0015]FIG. 4E shows a fifth embodiment and method of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,process steps, or equipment, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

[0017] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an assay”, includes more than one assay,reference to a “matrix” includes a plurality of matrixes and the like.

[0018] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0019] The term “acceptor” shall refer to any molecule or complexcapable of receiving energy emitting from a donor molecule or complex.Acceptor molecules may comprise any compounds whose absorption spectrumhas substantial overlap with the emission spectrum of the donormolecule. For instance, fluorescein, Cy5 and allophycocyanine could beused as acceptors for lanthanide; fast green and light green yellowishcould be efficient acceptors for most of the ruthenium complexes.

[0020] The term “donor” shall refer to any molecule or complex capableof emitting energy such that the emission spectrum substantiallyoverlaps the absorption spectrum of the acceptor.

[0021] The term “matrix” includes a complex or material capable ofsupporting other materials. This material may include gels, porousnetwork materials, organic and inorganic polymers, and biomaterials. Inparticular, the term refers to a complex made of sol-gel material.

[0022] The term “surface coating” refers to a thin film or layer appliedto a matrix material and shall include both known and unknownorgano-metallic compounds and elements capable of acting as energyacceptors. For purpose of this invention, the above term shall alsoinclude the situation when the donor is distributed in the entire matrixand parts thereof. In such a situation, at least a small fraction of thedonor near the surface of the matrix will participate in the energytransfer process.

[0023] The term “protection layer” refers to a material, film, layer, orcomplex that is transparent or translucent and may allow energy to passthrough it. In particular, this layer need not be completely contiguous,but it must be capable of preventing quencher molecules from reachingthe acceptor molecules of the surface coating.

[0024] The term “ligand” shall refer to any molecule, small molecule,protein, antibody, antigen or biomolecule capable of binding othermolecules. The term biolmolecule shall include proteins, DNAs, RNAs,polypeptides, and receptor molecules.

[0025] According to this invention a long lifetime donor such asruthenium tris diphenyl phenanthroline is sequestered or absorbed on asurface such as a silica particle. The surface with the absorbed donorlayer is then coated with a layer of optically transparent matrix suchas sol gel, which also acts as a diffusion barrier to quenchers such asoxygen. Thus, the donor molecule is trapped in a composite structure(referred to as a donor particle). The donor can be excited byappropriate radiation transmitted through the transparent barrier layer.Moreover, the transparent barrier allows fluorescence emission from thedonor to radiate through it.

[0026] The donor surface can be functionalized to immobilize arecognition element such as an antibody that complexes with the analyteof interest. Such an antibody tagged with a donor molecule can be usedin competitive immunoassays in a few different ways discussed below.

Construction of the Encapsulation Vesicles EXAMPLE 1

[0027] Ruthenium tris disphenyl phenanthroline was purchased from GFMChemicals (Catalog No. 2355, Lot # L027182). Silica (CAB-0-sil, TS-720)or hydrophobic amorphous fumed silica, was purchased from Cabotcorporation. Approximately 20 ml's of saturated chloroform solution ofthe ruthenium complex was prepared. 0.5 grams of hydrophobic silica wasadded to this dye solution. The mixture was then stirred forapproximately four hours until it appeared to be homogenous. Next,filtration was carried out and the silica particles were then washedwith acetone. Washing need not be completely efficient at this stepsince the filtration rate was extremely slow while the evaporation ofthe solvent was relatively fast. The dried silica particles were thenground.

[0028] The ruthenium encapsulated silica beads were put in a vial and 1ml of sol solution of the following composition was added: 3.0 mltetramethoxy silane, 1.2 ml water, 2 ml methanol and 0.4 ml 0.1 Nhydrochloroic acid. The mixture was stirred and substantially agitatedfor around four hours, until a viscous solution was reached. The productwas then transferred onto filtration paper and allowed to dry underambient conditions. The final dried produce was then ground and stored.

EXAMPLE 2

[0029] 0.5 grams of fumed hydrophobic silica (from Cabot) was suspendedin saturated solution of Ru(Ph₂Phen)₃ 2BPh₄ in 4 ml chloroform and 8 mlacetone. The mixtures were well shaken and let to stand at roomtemperature for a day. The silica particles were centrifuged and washedonce with methanol. Then 1 ml, sol-gel solution of the followingcomposition was added: 3.0 ml tetramethoxy silane, 1.2 ml water, 2.0 mlmethanol and 0.4 ml 0.1 N hydrochoric acid. The mixture was vortexed for15 minutes until it became homogenous and then the final product wascentrifuged at higher speed for 8 minutes. The supernatant wasdiscarded. The sol-gel protected fluorophore particle was allowed to dryunder ambient conditions and then ground.

[0030]FIG. 1 shows the composition of the present invention. Theinvention includes an encapsulation vesicle comprising a matrix 1, asurface coating 2, and a protection layer 3. The protection layer 3 hasan exterior surface 8 that may include one or more surface modificationssuch as a biomolecule. Biomolecules may include the use of an antibodyor similar type molecule capable of binding other molecules such as anantigen, or other molecules or proteins with an acceptor molecule.

[0031] The matrix 1 may comprise a variety of different materials thatare capable of supporting other compounds or materials. The matrix maycomprise a hydrophobic material. However, the material must be capableof being modified and or coated by a surface coating 2. Matrix 1, maycomprise a silica, sephadex or synthetic polymer type material capableof absorption of solvents. In its preferred embodiment, the material isa sol-gel composition. The sol-gel composition comprises a silica andsynthetic polymer composition. The surface of the matrix can be modifiedwith carboxyl and/or amino groups so that the organometallic complexescan be covalently attached. In other words, the surface is modified sothat long lifetime fluorophores can be absorbed or covalently linked onthe surface of the matrix.

[0032] Surface coating 2, may comprise a variety of materials includingorganic and inorganic molecules. Surface coating 2 must be capable ofacting as a donor. In its preferred embodiment, the surface coating 2comprises a ligand such as an organo-metallic material capable of actingas a donor molecule to a nearby energy acceptor. Organo-metallicmaterials are particularly effective as energy transfer donors becauseof their larger stoke shifts and high sensitivity. The organo-metallicmaterials that are used as donor molecules include ruthenium trisdiphenyl phenanthroline complexes. In particular, the rutheniumcomplexes have maximum emission peaks or spectra at about 650 nm.Europium complexes emit at about 615 nm and terbium complexes emit atabout 520 nm. A number of molecules can be used as donors. For instance,

[0033] In this complex, the coordination metal may include Ru, Os andRe. These also include materials that can form long lifetimemetal-ligand complexes with bipyridine and/or phenanthroline ligands.There are twelve different ligands designated R₁-R₁₂. These ligandsrepresent H, aryl, alkyl and aryl leading to the formation ofnon-substituted and substituted phenanthroline. Other donor moleculesinclude:

[0034] where R₁, R₂, R₃, R₄, represent H, alkyl, aryl, aryl leading tothe formation of non-substituted or substituted phenanthroline.

[0035] R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ represent H, alkyl, aryl, arylleading to the formation of ortho-aromatic phosphines

[0036] These fluorophores have a lifetime of greater than 100 ns and arethen susceptible to oxygen quenching.

[0037] Other long lifetime probes (donors) are lanthanides. Theseinclude the complexes of Eu, Th, Sm, Dy. Some examples of complexes withtypical ligands are shown below. They can be classified into 3 majorgroups including: 1. alkyl polycarboxyl complexes with at least oneshorter wavelength absorption antenna; 2. polypyridine complexes havingligands with carboxyl groups on or near the coordination site; and 3.polypyridine cryptate complexes. Chemical structures of each are shownfor 1-3 (from top to bottom) below.

[0038] wherein Ln=Eu, Th, Sm, Dy, and R represents H or a functionalitycapable of covalently linking to a matrix.

[0039] Acceptor molecules are different from donors in both compositionand function. For instance, the acceptor molecules or complexes cancomprise a ligand with a biomolecule. However, it should be noted thatthe only theoretical requirement is that the acceptor be positioned onor near the outside of the protection layer 3, discussed below, and thatthe acceptor's absorption band have a fair overlap with the emittingband of the donor. For example, Fast Green and Light Green SF yellowishand others can be used as acceptors for ruthenium complexes.Fluorescein, Cy5 and APC (allophycocyanin, a 105 kD phycobiliprotein)could be used as acceptors for lanthanide probes.

[0040] The protection layer 3 is important to the invention because itprevents the donor molecules on the surface coating 2 from beingeffected by ubiquitous quencher molecules such as oxygen. The mostproblematic quencher molecules being ubiquitous quenchers that exist inthe environment of the acceptor molecules. The protection layer 3 maycomprise any number of materials that are capable of excluding smallmolecule that may cause an effect on the quantum yield of the donormolecules. For instance, the actual protection layer 3 may comprise anynumber of materials capable of excluding small molecules, yet allow thetransfer of energy through the material. To prevent oxygen fromdiffusing, the surface has to be modified with hydrophilicfunctionalities, such as hydroxy, carboxy, and protonated amines.

[0041] In most cases, this means that the material must be translucentor transparent. It is important that the material be designed in such away that energy transfer can take place through or in conjunction withthe protection layer 3. The protection layer could be sol-gel (silica)and synthetic polymers. To prevent oxygen from diffusing the surface canbe modified with hydrophilic functionalities such as hydroxyl, carboxyl,or protonated amines (Zhou, Y. et al. 1996). The protection layer may beformed by the method of suspension polymerization. Protection layer 3may also contain an antibody or acceptor molecule on its exteriorsurface. The antibody is capable of binding other small molecules or anantigen. The Antigen may contain an acceptor molecule that is capable ofreceiving energy from the donor molecule that is on the surface coating2 inside the protection layer 3. The acceptor molecule and the donormolecule must be sufficiently close in space that the energy transfercan take place from the donor molecule to the acceptor molecule. Forinstance, if the donor molecule and the acceptor molecule are too farapart then energy transfer will either not take place, or will not beefficient enough to result in the facile measurement. The rate of energytransfer has a 1/r⁶ dependency on distance. The energy transfer will notbe significant or possible if the distance between the donor andacceptor is larger than 60-70 angstroms.

[0042] The method used in making the encapsulation vesicle isstraight-forward. In the first step of the invention the matrix isprepared using the silica particles. Next, a monolayer of thefluorescent dye is added to the particles. The layer is allowed to dryand the protection layer is added to the surface coating.

[0043]FIG. 2 shows a diagram of a second embodiment of the compositionof matter of the present invention. The encapsulation vesicle 5 has theacceptor molecules 4 attached directly to the protection layer 3. Theencapsulation vesicle 5 is designed in such a way that the acceptormolecules 4 may receive energy or electrons from the surface coating 2that contains the donor molecules. This invention provides for ease inmonitoring changes in the encapsulation vesicle(s) over time. FIG. 2shows the simplest case scenario where the acceptor molecule 4 isdirectly attached to the protection layer 3. However, acceptor molecules4 need not be attached directly to protection layer 3 and in some casesmay actually be free in solution.

[0044]FIG. 3 shows a third embodiment of the composition of matter ofthe present invention. The figure shows the encapsulation vesicle 5 onone end of a substrate 6 and the relationship between the acceptormolecule(s) 4 and the encapsulation vesicles 5. The embodiment includesthe use of an enzyme or enzyme action that results in cleavage of thesubstrate 6 and separation of the encapsulation vesicle 5 from theacceptor or acceptor molecules 4. Cleavage is accomplished by the enzymeand energy transfer is no longer possible between the donor andacceptor.

[0045] The method of the present invention (illustrated in FIGS. 4A-4D)is capable of determining and quantifying binding reactions used inimmunoassays. In the present method the reactants are labeled with aphotoluminescent energy transfer donor and acceptor. The inventiondiffers in that there is a protection step included so as to limit oreradicate the effects of collisional quenchers on the donor complex ormolecule. The immunoassay or immuno-reaction brings the donor andacceptor molecules into close proximity. This is important, because whenthe reaction product is excited through application of an externalsource, energy transfer can occur between the donor and the acceptor.The present method can be used with a variety of different assaysincluding competitive and non-competitive. In addition, the antigenand/or antibody can be labeled with more than one acceptor molecules.

[0046] The method of the present invention includes mixing a firstbinding molecule with a second binding molecule. The second bindingmolecule may be free in solution or fixed to a vesicle surface. In otherwords, the donor and the acceptor are brought into close interactingproximity, and are capable of producing a detectable luminescencelifetime change in the photoluminescence lifetime of the donor. Themethod also includes the steps of encapsulating the donor molecule sothat collisional quenchers such as oxygen will not interfere with energytransfer from the donor to the acceptor, exposing the sample to anexciting amount of radiation and then detecting the resulting emission.Lastly, the apparent luminescent lifetime of the donor is calculated toquantify the binding of the first binding molecule to the second bindingmolecule.

EXAMPLE 1

[0047] In a first embodiment of the invention (See FIG. 4A), a knownconcentration of analyte (the antigen) of interest, tagged with anacceptor, is incubated with the donor-tagged antibody described above,thus allowing the antibody-antigen immuno-complex to form. After thisstep is complete, the fluorescence lifetime and/or intensity offluorescence emitted by the donor, in response to appropriateexcitation, are measured. Subsequently, sample is introduced to theabove immuno-complexes. Upon appropriate conditioning, the antigen ofinterest will displace the tagged antigen from the immuno-complex. Thenumber of such displacement events will be proportional to the ratio ofthe concentration of the antigen in the sample. This will decreaseimmuno-complexes wherein the donor to acceptor energy transfer occurs.This will result in a net decrease in the lifetime and/or intensity ofthe donor emission. This increase in lifetime and/or intensity isrelated to the concentration of the analyte.

[0048] The acceptor may be any molecule whose absorption characteristicssignificantly overlap the emission characteristics of the donor.Selection of the acceptor and the corresponding donor is primarily basedon the criteria of overlapping emission-absorption characteristics andthe efficiency of the resultant energy transfer. The practice ofteaching in several embodiments would be obvious to those familiar withthe field of assay design such as fluorometry and fluorophore chemistry.

[0049]FIG. 4A shows the method of the present invention using standardimmunoassay components. In the method, an antibody 7 is linked to theprotection layer 3 that encapsulates the surface coating 2 on matrix 1.The analyte 8 is attached to the acceptor molecule 4. The acceptormolecule 4 receives the energy transfer from the surface coating 2. Thisis shown in the diagram by an arrow. The immunoassay is designed in away to detect that presence and quantity of the analyte 8 the may existin solution. The invention has the ability to improve both intensity andlifetime fluorescence measurements.

EXAMPLE 2

[0050] In an alternative embodiment, after incubation with the sample toform the donor-tagged antibody analyte immuno-complex, anacceptor-tagged antibody is introduced to form a sandwich complex (SeeFIG. 4B). The methods and chemistries for the sandwich complex formationare well known in the art of immunochemistry. The second antibody,tagged with the acceptor molecule, presents the acceptor in such a waythat the fluorescence emitted by the donor is quenched. The number ofquenching events is proportional to the analyte concentration. Thus, thelifetime and intensity of the fluorescence decreases in proportion tothe amount of analyte in the sample.

[0051]FIG. 4B shows a second method and embodiment of the inventionexcept with a sandwich assay or immunoassay. In this case the existenceand quantity of the analyte 8 is determined by the binding of theantibody 7′ with the acceptor 4 and the antibody 7 with the analyte 8.In other words, the analyte is sandwiched between the antibodies 7 and7′. Energy is transferred from the surface coating 2 to the acceptor 4attached to the antibody 7′ that has become bound to the analyte 8.

EXAMPLE 3

[0052] Each of the above embodiments of the invention teachesapplications with immunoassays. However, this invention can be appliedto a number of different assays. These assays include DNA hybridizationdetection, detection of any receptor binding to its complement, anddetection of cleavage of a bond linking a group carrying the acceptor toa group carrying a donor etc. For example, one could design assays fordetection of an inclusion compound complexing with a receptor molecule.In another embodiment this invention can be practiced for determiningactivity of a protease molecule. For example a substrate molecule can betagged with the acceptor in a way that there is net energy transfer fromthe donor to the acceptor. Now if a protease molecule cleaves a site onthe substrate somewhere between the acceptor and the donor, the two tagswill be separated substantially and there will be no net energytransfer. Such a scheme can be used to determine the activity of aprotease molecule at intervening sites on the substrate (See FIG. 3 foran illustration). Other embodiments could also include the use of themethod with DNA or RNA as a probe or with aptamers for protein binding.These novel methods are discussed in more detail below.

[0053]FIG. 4C shows another embodiment of the invention except in thiscase an RNA or DNA probe is used. The diagram depicts a situation wherenot all the nucleotides base pair. The invention, however, includes thesituation when all the nucleotides of the probe and target base pair aswell as partial base pair. The DNA or RNA probe 9 is attached to theprotection layer 3. The DNA or RNA probe 9 can be a known or unknownsequence and can be used to bind other nucleotides 11 that have beencovalently attached to the acceptor molecule 4. This type of assay canthen be used to effectively monitor the quantity of nucleotides 11 thatare free in solution or bound to the DNA or RNA probe 9. The arrow inthe diagram shows how the energy can be transferred from the surfacecoating 2 to the acceptor 4. This embodiment or similar type embodimentshave the capability of being applied to a variety of technologiesincluding micro-arrays.

[0054]FIG. 4D shows another embodiment of the present invention. In thiscase, an aptamer 13 is attached to the protection surface 3 that iscapable of binding a protein 15 with an attached acceptor molecule 4.When the protein 15 contacts the aptamer 13 it is bound so that theacceptor molecule 4 is in close proximity to the surface coating 2. Thearrow in the diagram shows how energy transfer takes place from thesurface coating 2 to the acceptor 4 that is bound to the protein 15.This embodiment or similar type embodiments have the capability of beingapplied to a variety of technologies including micro-arrays.

[0055]FIG. 4E shows an embodiment similar to the 4A embodiment. However,excess analyte 8 is present in solution. The analyte 8 with boundacceptor 4 competes for binding to the antibody 7 with the analtye 8without bound acceptor. Lower energy transfer results when less analytewith bound acceptor binds to the antibody 7.

[0056] A number of photoluminescent donors may be used in the method ofthe invention and include the compounds listed above as well as groupssuch as cyanines, oxazines, thiazines, porphyrins, phthalocyanines,fluorescent infrared-emitting polynuclear aromatic hydrocarbons,phycobiliproteins, squaraines and organo-metallic complexes.

[0057] In the method, a number of important acceptors may be used inconjunction with the acceptors described above for the composition. Forinstance, some of the photoluminescent acceptors may also include thecyanines, oxazines, thiazines, porphyrins, phthalocyanines, fluorescentinfrared-emitting polynuclear aromatic hydrocarbons, phycobiliproteins,squaraines, organo-metallic complexes, and azo dyes.

REFERENCES

[0058] U.S. Pat. No. 5,631,169, granted May 20, 1997, by Lakowicz, etal.

[0059] Jiyan Chen and Paul R. Selvin, “Thiol-Reactive LuminescentChelates of Terbium and Europium”, Bioconjugate Chem., 1999, 10,311-315.

[0060] Gerard Mathis, “Rare Earth Cryptates and HomogenousFluorimmunoassays with Human Sera”, Clin. Chem., 39(9), 1993, 1953-1959.

[0061] Gerard Mathis, “Probing Molecular Interactions with HomogenousTechniques Based on Rare Earth Cryptates and Fluorescence EnergyTransfer”, Clin. Chem. 41(9), 1995, 1391-1397.

[0062] P. Aich, et al., “M-DNA: A Complex Between Divalent Metal Ionsand DNA which Behaves as a Molecular Wire”, J. Mol. Biol 1999, 294,477-485.

[0063] Y. Zhou, et al., “Preparation of Hyperbranched Polymer FilmsGrafted on Self-Assembled Monolayers”, J. Am. Chem. Soc. 118 (1996)3773-3774.

[0064] Ilkka Hemmila, et al., “Time-resolved Fluorometry: An Overview ofthe Labels and Core Technologies for Drug Screening Applications”, DDTVol. 2, No. 9, Sept. 1997, 373-381.

[0065] Edward M. Kober, et al., “Synthetic Control of Excited States.Nonchromophoric Ligand Variations in Polypyridyl Complexes of Osmium(II)”. Inorg. Chem. 1985, 24, 2755-2763.

We claim:
 1. An encapsulation vesicle for an assay, comprising: (a) amatrix having a surface; (b) a surface coating on said matrix; (c) aprotection layer encapsulating said surface coating for protecting saidsurface coating from a quencher molecule; and (d) a ligand attached tosaid protection layer.
 2. An encapsulation vesicle as recited in claim1, wherein said matrix is a sol gel material.
 3. An encapsulationvesicle as recited in claim 1, wherein said matrix surface comprisessilica and synthetic polymer.
 4. An encapsulation vesicle as recited inclaim 1, wherein the matrix surface is modified with carboxyl groups sothat organometallic complexes can be covalently attached to the surface.5. An encapsulation vesicle as recited in claim 1, wherein the matrixsurface is modified with amino groups so that organometallic complexescan be covalently attached to the surface.
 6. An encapsulation vesicleas recited in claim 3, wherein the matrix is modified so that longlifetime fluorophores can be either absorbed or covalently linked to thematrix.
 7. An encapsulation vesicle as recited in claim 1, wherein saidsurface coating comprises at least one donor molecule.
 8. Anencapsulation vesicle as recited in claim 7, wherein said donor moleculeis an organometallic material.
 9. An encapsulation vesicle as recited inclaim 8, wherein said donor molecule is:

where M is s selected from the group consisting of Ru, Os and Re; R₁ isselected from the group consisting of H, alkyl, aryl, and aryls leadingto a non-substituted or substituted phenanthroline; R₂ is selected fromthe group consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₃ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₄ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₅ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₆ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₇ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₈ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₉ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₁₀ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; R₁₁ is selected from thegroup consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline; and R₁₂ is selected fromthe group consisting of H, alkyl, aryl, and aryls leading to anon-substituted or substituted phenanthroline.
 10. An encapsulationvesicle as recited in claim 8, wherein said organometallic material is aruthenium tris diphenyl phenanthroline complex.
 11. An encapsulationvesicle as recited in claim 8, wherein said organometallic material hasan emission at about 650 nm.
 12. An encapsulation vesicle as recited inclaim 8, wherein the donor molecule is selected from the groupconsisting of:

where R₁, R₂, R₃, R₄, represent H, alkyl, aryl, aryl leading to theformation of non-substituted or substituted phenanthroline; and whereinR₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ represent H, alkyl, aryl, aryl leadingto the formation of ortho-aromatic phosphines.
 13. An encapsulationvesicle as recited in claim 8, wherein said donor molecule is selectedfrom the group consisting of:

where Ln=Eu, Th, Sm, Dy, and R represents H or a functionality capableof covalently linking to a matrix.
 14. An encapsulation vesicle asrecited in claim 8, wherein the donor molecule is any molecule having afluorescence lifetime greater than 100 nanoseconds and the molecules aresusceptible to quenching by oxygen.
 15. An encapsulation vesicle asrecited in claim 2, wherein said protection layer comprises atranslucent material.
 16. An encapsulation vesicle as recited in claim2, wherein said protection layer comprises a transparent material. 17.An encapsulation vesicle as recited in claim 2, wherein said protectionlayer comprises sol-gel (silica) and synthetic polymers.
 18. Anencapsulation vesicle as recited in claim 2, wherein said protectionlayer is modified with hydrophilic functionalities selected from thegroup consisting of hydroxyl, carboxyl and protonated amines.
 19. Anencapsulation vesicle as recited in claim 2 that was formed bysuspension polymerization.
 20. An encapsulation vesicle as recited inclaim 1, wherein said ligand attached to said protection layer furthercomprises an acceptor molecule capable of receiving energy transfer fromsaid donor molecule of said surface coating.
 21. An encapsulationvesicle as recited in claim 1, wherein said assay is an immunoassay. 22.An encapsulation vesicle as recited in claim 3, further comprising aligand attached to the protection layer and having an acceptor moleculecapable of receiving energy transfer from a donor molecule.
 23. Anencapsulation vesicle as recited in claim 1, wherein the acceptor'sabsorption band overlaps with the emission band of the donor.
 24. Anencapsulation vesicle as recited in claim 1, wherein the acceptor isselected from the group consisting of fluorescein, Cy5 andallophycocyanin.
 25. An encapsulation vesicle as recited in claim 1,wherein said ligand is an antibody.
 26. An encapulation vesicle asrecited in claim 1, wherein said assay is a sandwich assay.
 27. Anencapsulation vesicle as recited in claim 1, wherein the biomolecule isselected from the group consisting of proteins, DNA, RNA, polypeptides,aptamers and receptor molecules.
 28. A method of quantifying an analytein a sample, comprising the steps of: (a) mixing a first bindingmolecule with a second binding molecule, wherein the first bindingmolecule competes with an analyte for binding the second bindingmolecule, wherein one of the first and second binding molecules islabeled with a photoluminescent energy transfer donor and the other islabeled with a photoluminescent energy transfer acceptor, wherein thephotoluminescent energy transfer donor and acceptor are chosen such thatwhen the first binding molecule binds to the second binding molecule,the donor and acceptor are brought into intreracting proximity,producing a detectable luminescence change in the donor; (b)encapsulating a second binding molecule; (c) exposing the sample to anexciting amount of radiation; (d) detecting the resulting emission; and(e) calculating the apparent luminescence of the donor to quantifybinding of the first binding molecule to the second binding molecule andthereby inversely quantifying the analyte.
 29. The method of claim 29,wherein the photoluminescent donor is selected from the group consistingof cyanines, oxazines, thiazines, porphyrins, phthalocyanines,fluorescent infrared-emitting polynuclear aromoatic hydrocarbons,phycobiliproteins, squaraines and organo-metallic complexes.
 30. Themethod of claim 29, where the photoluminescent acceptor is selected fromthe group consisting of cyanines, oxazines, thiazines, porphyrins,phthalocyanines, polynuclear aromatic hydrocarbons, phycobiliproteins,squaraines, organo-metallic complexes, and azo dyes.
 31. The sandwichmethod of quantifying an analyte in a sample, comprising the steps of:(a) mixing a first binding molecule with a second binding molecule,wherein the first binding molecule competes with an analyte for bindingthe second binding molecule, wherein one of the first and second bindingmolecules is labeled with a photoluminescent energy transfer donor andthe other is labeled with a photoluminescent energy transfer acceptor,wherein the photoluminescent energy transfer donor and acceptor arechosen such that when the first binding molecule binds to the secondbinding molecule, the donor and acceptor are brought into intreractingproximity, producing a detectable luminescence lifetime change in thephotoluminescence lifetime of the donor; (b) encapsulating a secondbinding molecule; (c) exposing the sample to an exciting amount ofradiation; (d) detecting the resulting emission; and (e) calculating theapparent luminescence lifetime of the donor without the use of afluorescence intensity measurement to quantify the immune complex,thereby quantifying the analyte.